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A complementary DNA (cDNA) was designed to simultaneously hybridize with the ochratoxin A (OTA) aptamer and the fumonisin B1 (FB1) aptamer to form a unique Y-shaped DNA structure and to achieve simultaneous detection. Gold nanorods (AuNRs) were used to immobilize thionine (Th), thiolated ferrocene (Fc), thiolated OTA aptamer (Apt1), and thiolated FB1 aptamer (Apt2), to form an amplified signal element and a recognition element. The Apt1-AuNRs-Th complex and the Apt2-AuNRs-Fc complex hybridize with cDNA to form a unique Y-DNA structure on a gold electrode. This produces two initial electrochemical signals [with 177 μΑ cm−2 near −0.2 V, and 3121 μΑ cm−2 near +0.46 V (vs. Ag/AgCl)] by differential pulse voltammetry. Upon addition of 0.1 ng mL−1 OTA and 0.1 ng mL−1 FB1, the aptamers bind the two toxins. This results in the release of Apt1-AuNRs-Th and Apt2-AuNRs-Fc, so the peak currents densities decrease to 115 μΑ cm−2 and 209 μΑ cm−2. The assay allows simultaneous determination of OTA and FB1 in the 1.0 pg·mL−1 to 100 ng·mL−1 concentration ranges, with LODs of 0.47 and 0.26 pg·mL−1. The assay is reproducible, stable and specific. It was applied to the determination of OTA and FB1 in spiked beer, with recoveries between 89.0% and 102.0%. [ABSTRACT FROM AUTHOR]